Protein kinases are involved in the regulation of cellular metabolism, proliferation, differentiation and survival. Protein kinases phosphorylate proteins on serine/threonine or tyrosine residues. Activation of one class of kinase typically leads to activation of more than one signaling pathway through signaling crosstalk. The receptor tyrosine kinases (RTKs) are a major type of cell-surface receptors, where the intracellular part of the receptor has a kinase domain. The activating ligands are peptide/protein hormones, like the FL-ligand, Vascular Endothelial Growth factor (VEGF), Epidermal Growth factor (EGF), Fibroblast growth factor (FGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), insulin, etc. Binding of a ligand to the extracellular domain of an RTK results in receptor dimerisation and a conformational change that activates the kinase site on the intracellular domain. The kinase activity leads to a signal-transduction cascade by phosphorylation of other proteins that regulates cellular physiology and patterns of gene expression (for a review see Schlessinger, J. (2000) Cell 103: 211-225; and Blume-Jensen P. & Hunter T. (2001) Nature 411: 355-365). The intracellular signaling proteins activated in the signaling cascade can be other kinases and/or proteins involved in transcription and translation. There are several families of intracellular kinases. The Janus kinase (JAK) family of tyrosine kinases (JAK1, 2, 3, and Thy1) are activated through interaction with other proteins (see O'Shea, J. J. et al. (2002) Cell 109 (Suppl.) 121-131 and references therein). Serine/threonine kinases like the protein kinase C (PKC) family of isozymes and the mitogen activated kinases (MAP-kinase family) are also involved in the regulation of cell survival, proliferation and differentiation. The PKC-isozymes are activated by calcium, and diacylglycerol is an allosteric activator of some of the members of the PKC family (alpha beta gamma). Intracellular kinases interact with other proteins and are often translocated to other compartments upon activation (see Manning, G. et al. (2002) Science 298: 1912-1934; Martin. P. M. & Hussaini I. M. (2005) Expert Opin. Ther. Targets 9(2) 299-313 and references therein). Membrane association can be regulated by myristoylation, as in the case of PKC isozymes. Nuclear association has been described for several different classes of kinases. MAP-kinases are activated by other proteins and capable of translocating to the nucleus, where proteins involved in transcription and regulators of cell-cycle and differentiation becomes phosphorylated.
During normal development and differentiation both kinase activation and deactivation is tightly regulated. Oncogenic mutations, leading to constitutively active kinases, can trans-form normal cells to cancer cells. An activating mutation can be the result of a chromosome translocation giving rise to a fusion protein, for example as in chronic myeloic leukemia where the ABL-tyrosine kinase domain is fused to the BCR protein (for a review see Östman, A. (2007) Helix Review Series Oncology 2: 2-9; and Deininger, M. et al. (2005) Blood 105: 2640-2653).
During normal hematopoesis, FLT3 is active at the myeloblast stage, but the FLT3 activity is then switched off upon normal hematopoetic differentiation to mature blood cells (Gilliand, D. G. & Griffin, J. D. (2002) Blood 100: 1532-1542; Weisel, K. C. et al. (2007) Ann. N.Y. Acad. Sci. 1106: 190-196). In acute myeloic leukemia, (AML), the FLT3 expression is high in the majority of patients (70-90%) (Carow, C. E. et al. (1996) Blood 87 (3): 1089-1096; and Rosnet, O. et al (1993) Crit. Rev. Oncogenesis 4: 595-613). Furthermore, the FLT3 kinase activity is upregulated in one third of the patients due to an internal tandem duplication in the juxtamembrane position (FLT3-ITD), resulting in a ligand independent receptor dimerization and a constitutively active kinase. FLT3-ITD is a prognostic marker, with a statistically significant reduction in survival in the patient population harboring the mutation, specially if both alleles are affected. There are also activating point mutations (FLT3-PM) of FLT3 described in AML patients. These activating mutations can be found in the activation loop of the kinase domain (AL-mutations) or in the juxtamembrane domain (JM-mutations). For a review see Carow, C. E. et al. (1996) Blood 87 (3): 1089-1096; Tickenbrock, L. et al. (2006) Expert Opin. Emerging Drugs 11(1): 153-165; Anjali S. & Advani, A. S. (2005) Current Pharmaceutical Design 11: 3449-3457; Lee B. H. et al. (2007) Cancer Cell 12: 367-380); Stam, R. W. et al. (2005) Blood 106(7): 2484-2490; and references therein. In addition FLT3-ITD or FLT3-PM has been found in subsets of patients with other lymphoid or myeloid malignancies such as MLL, T-ALL and CMML, and a high FLT3-activity has been described in B-ALL (for a review see Lee, B. H. et al. (2007) Cancer Cell 12: 367-380.
However, FLT3 activity is part of the normal hematopoesis. If the proliferation of immature blast cells in the bone marrow is dysregulated, by an overstimulation of kinases like FLT3, this might result in a depletion of other hematopoetic cells. Blast cells then enter the bloodstream, instead of mature differentiated cells. The acute leukemic state results in anemia and neutropenia. Thus, blocking unfavorable kinase activity could reduce the proliferation of blast cells, and reduce the leukemic state. Several FLT3 kinase inhibitor has been tested in models of AML and in clinical indications where FLT3 is involved (Cheng, Y. & Paz, K. (2008) IDrugs 11(1): 46-56; Kiyoi, H. et al. (2007) Clin. Cancer Res. 13(15): 4575-4582; Roboz, G. J. et al. (2006) Leukemia 20: 952-957; Tse, K-F. et al. (2002) Leukemia 16: 2027-2036; Smith, B. D. et al. (2004) Blood 103: 3669-3676; Knapper, S. et al. (2006) Blood 108 (10): 3494-3503; and Furukawa, Y. et al. (2007) Leukemia 21: 1005-1014). The AML cell-line MV4-11 carries the FLT3-ITD. This cell-line is very sensitive in viability/proliferation assays to inhibitors of FLT3 activity. However, in ex-vivo patient cells there is also crosstalk between the signaling pathways, molecules activated downstream of the FLT3 receptor can also be activated by other kinases. Knapper et al 2006 showed that even though the autophosphorylation of FLT3 was down-regulated in patient cells after exposure to FLT3 inhibitors, the phosphorylation state of the down-stream effectors STAT and ERK were not diminished, possibly due to dysregulation of other signaling pathways apart from FLT3-phosphorylation.
The activity of FLT3 and other RTK is regulated by autophosphorylation and internalisation, the phosphorylation of the receptor is then removed by specific phosphatases that are also subject to regulation. A dysregulation of the internalization process and the dephosphorylation of the phosphatases could also have an impact on the RTK-activity and thus alter viability and proliferation of cells. As there are several orders of regulation, a kinase inhibitor needs to have a certain profile regarding its target specificity and mode of action to effectively inhibit proliferation and viability in cancer or a proliferative disorder.